A ratiometric fluorescence probe based on carbon dots for discriminative and highly sensitive detection of acetylcholinesterase and butyrylcholinesterase in human whole blood

Article abstract of DOI:10.1016/j.bios.2019.02.031

A ratiometric fluorescence probe based on carbon dots (CDs) was developed for discriminative and highly sensitive detection of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in human whole blood. When o-phenylenediamine (OPD) was oxidized by Cu²⁺, the product 2,3-diaminophenazine (oxOPD) could effectively quench the fluorescence of CDs at 460 nm due to the inner filter effect and gave rise to a new emission peak at 570 nm. The AChE or BChE catalyzed hydrolysis reaction of acetylthiocholine or butyrylthiocholine to generate thiocholine, whose sulfhydryl group strongly captured Cu²⁺ to inhibit the oxidization of OPD, thus effectively preserving the natural fluorescence emission of CDs. The resulting fluorescence intensity ratio served as the signal output of the probe for cholinesterases (ChEs) activity sensing. The activities of AChE and BChE were determined to range from 0.2 to 14.0 U L^?1 and from 0.1 to 5.0 U L^?1, with detection limits of 0.1 U L^?1 and 0.04 U L^?1, respectively. Additionally, the IC₅₀ of tacrine and ethopropazine for the inhibition of AChE and BChE were estimated to be 29.8 nM and 132.6 nM, respectively. Moreover, the probe was successfully applied to the discriminative determination of AChE and BChE in human whole blood without any pretreatment. These results suggested that the proposed strategy provided a discriminative, sensitive and robust analytical platform for ChEs clinical diagnostics and drug screening.

Full text of DOI:10.1016/j.bios.2019.02.031

Author’s Accepted Manuscript
A ratiometric fluorescence probe based on carbon
dots for discriminative and highly sensitive
detection
of
acetylcholinesterase
and
butyrylcholinesterase in human whole blood
Xiaoman Xu, Yao Cen, Guanhong Xu, Fangdi
Wei, Menglan Shi, Qin Hu
www.elsevier.com/locate/bios
PII:
S0956-5663(19)30138-1
https://doi.org/10.1016/j.bios.2019.02.031
BIOS11127
DOI:
Reference:
To appear in: Biosensors and Bioelectronic
Received date: 17 November 2018
Revised date: 3 February 2019
Accepted date: 4 February 2019
Cite this article as: Xiaoman Xu, Yao Cen, Guanhong Xu, Fangdi Wei, Menglan
Shi and Qin Hu, A ratiometric fluorescence probe based on carbon dots for
discriminative and highly sensitive detection of acetylcholinesterase and
butyrylcholinesterase in human whole blood, Biosensors and Bioelectronic,
https://doi.org/10.1016/j.bios.2019.02.031
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A ratiometric fluorescence probe based on carbon dots for
discriminative and highly sensitive detection of acetylcholinesterase
and butyrylcholinesterase in human whole blood
1
1,
Xiaoman Xu , Yao Cen *, Guanhong Xu, Fangdi Wei, Menglan Shi, Qin Hu*
School of Pharmacy, Nanjing Medical University, Nanjing, 211166, P. R. China
*
Corresponding Authors.
E-mail addresses: yaocen@njmu.edu.cn; huqin@njmu.edu.cn. Tel/Fax: +86-25-
8
6868468.
1
These authors contributed equally to this work.
Abstract
A ratiometric fluorescence probe based on carbon dots (CDs) was developed for
discriminative and highly sensitive detection of acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) activity in human whole blood. When o-
2
+
phenylenediamine (OPD) was oxidized by Cu , the product 2,3-diaminophenazine
oxOPD) could effectively quench the fluorescence of CDs at 460 nm due to the inner
(
filter effect and gave rise to a new emission peak at 570 nm. The AChE or BChE
catalyzed hydrolysis reaction of acetylthiocholine or butyrylthiocholine to generate
2
+
thiocholine, whose sulfhydryl group strongly captured Cu to inhibit the oxidization
of OPD, thus effectively preserving the natural fluorescence emission of CDs. The
resulting fluorescence intensity ratio served as the signal output of the probe for
cholinesterases (ChEs) activity sensing. The activities of AChE and BChE were
-1
-1
determined to range from 0.2 to 14.0 U L and from 0.1 to 5.0 U L , with detection
1

-1
-1
limits of 0.1 U L and 0.04 U L , respectively. Additionally, the IC of tacrine and
5
0
ethopropazine for the inhibition of AChE and BChE were estimated to be 29.8 nM
and 132.6 nM, respectively. Moreover, the probe was successfully applied to the
discriminative determination of AChE and BChE in human whole blood without any
pretreatment. These results suggested that the proposed strategy provided a
discriminative, sensitive and robust analytical platform for ChEs clinical diagnostics
and drug screening.
Keywords: Acetylcholinesterase; Butyrylcholinesterase; Discriminative detection;
Carbon dots; Ratiometric fluorescence
1
. Introduction
Cholinesterases (ChEs) are critical enzymes in the central nervous system that
catalyze the hydrolysis reaction of choline esters to choline (Mikalsen et al., 1986).
There are two major ChE isoenzymes, namely acetylcholinesterase (AChE) which
mainly binds to the erythrocyte membrane, and butyrylcholinesterase (BChE) which
mainly presents in plasma (Miao et al., 2010). AChE plays vital roles in maintaining
the levels of the neurotransmitter acetylcholine (Soreq and Seidman, 2001).
Moreover, AChE and BChE activities are recognized as diagnostic biomarkers in
organophosphate poisoning, and nerve agent exposure (Santarpia et al., 2013). In this
regard, the development of discriminative and sensitive detection methods for both
AChE and BChE activities in whole blood is of significant importance.
Currently, the most widely used approach for ChEs activities is Ellman’s
colorimetric method (Ellman et al., 1961). However, the widespread application of
this method is limited by its shortcomings. For instance, its sensitivity is poor due to
2

the colorimetric method, the thiol groups in samples react with Ellman’s reagent to
introduce false-positive effect, and the massive Soret absorbance of hemoglobin
interferes with the absorbance of the reaction product, and whole blood samples need
laboring pretreatment (Miao et al., 2010). The choline oxidase coupled multienzyme
assay is an effective method for monitoring ChE activities (Gill et al., 2008);
unfortunately, the process requires time-consuming procedures. While there have
been several alternative methods, including mass spectrometry (de Jong et al., 2006),
and radiometry (Winteringham and Disney, 1962), but they generally suffer from the
requirement of special instruments or radiolabeling. Recently, several fluorescent
nanomaterials including semiconductor quantum dots (Saa et al., 2010), silver/gold
nanoclusters (Li et al., 2013), and carbon dots (Qian et al., 2016; Chen et al., 2018)
have been reported for ChEs activities sensing. However, such probes based on a
single fluorescent intensity change cannot avoid the interferences from probe
concentrations, light source, and measurement conditions. Importantly, all of these
methods are challenged by discriminatively determining both AChE and BChE
activities owing to the coexistence of AChE and BChE in whole blood and
overlapping substrate affinities.
Carbon dots (CDs) have received great attention in the field of biosensing,
bioimaging, and drug delivery due to their convenient preparation, multicolored
emissions and high biocompatibility (Lim et al., 2015; Baker and Baker, 2010). In
particularly, the strong photoluminescence property makes CDs an ideal choice for the
development of fluorescence biosensors. Recently, several groups have exploited
CDs-based fluorescence probe to detect metal ions, DNA, dopamine, and enzymes
(Dong et al., 2012; Noh et al., 2013; Qu et al., 2013; Li et al., 2016). However, these
biosensors exclusively depended on a single fluorescent intensity output, resulting in
3

low signal-to-background ratios. Alternatively, several CDs-based ratiometric
2
+
2+
fluorescence biosensors have also been constructed for the detection of Cu , Hg ,
and H S (Zhu et al., 2012; Zhao et al., 2017; Yu et al., 2013). However, to our
2
knowledge, the development of CDs-based probe for the ratiometric fluorescence
detection of AChE and BChE remains largely unexplored.
Herein, we developed a ratiometric fluorescence probe based on CDs for the
discriminative and highly sensitive detection of AChE and BChE in human whole
blood. As shown in Fig. 1A, the CDs prepared by (3-aminopropyl)triethoxysilane and
glycerol exhibited a strong fluorescence peak. o-Phenylenediamine (OPD) was
2
+
oxidized by Cu to generate 2,3-diaminophenazine (oxOPD), which not only
effectively quenched the fluorescence of CDs due to the inner filter effect (IFE), but
also yielded a new emission peak at 570 nm. In the presence of ChEs, the substrate
acetylthiocholine (ATCh) or butyrylthiocholine (BTCh) was hydrolyzed into
2
+
thiocholine (TCh), whose sulfhydryl group can strongly capture Cu , and the
oxidization of OPD was blocked, resulting in fluorescence changes of the system.
Therefore, the fluorescence recovery at the emission peak of 460 nm together with
fluorescence decrease at the emission peak of 570 nm occurred in the system,
allowing the development of ratiometric fluorescence strategy for ChEs activities
sensing. Importantly, as illustrated in Fig. 1B, by taking advantage of specific
substrates and a selective BChE inhibitor (ethopropazine, Etho), the probe could
directly detect AChE and BChE in human whole blood. On the basis of these features,
the developed probe allowed for the discriminative, simple, and sensitive detection of
ChEs activity with great potential for further clinical diagnosis and drug screening.
4

Fig. 1. Schematic illustration of the CDs-based ratiometric fluorescence probe for the
detection of AChE and BChE in human whole blood.
2
. Experimental
2
.1 Determination of AChE and BChE in aqueous solution
First, 10 μL of AChE or BChE with different activities and 0.4 mM ATCh or
BTCh were added to 100 μL of Tris-HCl buffer (pH 7.5) and incubated at 37 °C for
2
+
3
2
0 min. Then, 0.07 mM Cu and 0.5 mM OPD was added and incubated at 37 °C for
h. Afterwards, 20 μL of CDs was added, the solution was incubated for 20 min and
subjected to fluorescence measurements. The solutions were prepared by adding other
enzymes instead of AChE or BChE for the specificity assay.
2
.2 Determination of AChE and BChE in human whole blood
5

The preparation of blood samples is described in the Supporting Information.
The whole blood and erythrocytes were finally diluted 3000-fold for AChE
determination, while the whole blood and plasma were finally diluted 10000-fold for
BChE determination. For the AChE analysis, ATCh was used as the substrate and
Etho as the BChE inhibitor. For the BChE analysis, BTCh was used as the substrate.
The following procedures for the samples were similar to that described above.
3
. Results and discussion
3
.1 Characterization of CDs
The CDs were prepared through a facile hydrothermal method (Zou et al., 2016).
The transmission electron microscope (TEM, Fig. 2A) image showed that CDs were
monodispersed with a diameter of approximately 9.0 nm, which agreed with the
dynamic light scattering (Fig. S1). Compared with the FT-IR spectrum of pure
-1
APTES, CDs presented new absorption bands at 1650 cm (stretching vibration of
C=O), which indicated the dehydration and carbonization (Fig. S2). The X-ray
photoelectron spectroscopy survey scan verified the successful synthesis of CDs (Fig.
S3). The fluorescence emission of CDs (Fig. 2B) showed a red-shift with the
excitation wavelength in the range of 310 to 420 nm, indicating that CDs displayed an
excitation-dependent fluorescence behavior, which was a trademark property of CDs
(Huang et al., 2014). The fluorescence of CDs appeared strong blue upon UV light
excitation (Fig. 2B, inset) and the fluorescence intensity of CDs exhibited no
significant changes under different pH conditions (Fig. S4).
6

Fig. 2. (A) TEM image of CDs. Inset shows the high-resolution TEM image of CDs.
B) UV-vis absorption spectrum of CDs and fluorescence emission spectra of CDs at
(
different excitation wavelengths. Inset: photographs of CDs under visible and UV
light.
3
.2 Construction of CDs-based ratiometric probe
The fluorescence spectra of CDs exhibited no evident changes with the addition
2
+
of Cu and other metal ions (Fig. S5). To achieve a balance between the fluorescence
intensities of CDs and oxOPD, we used 380 nm as the excitation wavelength. The
2
+
colorless and nonfluorescent OPD could be oxidized by Cu , and the resultant
oxOPD showed an obvious absorption peak at 415 nm, as well as a strong
fluorescence emission peak at 570 nm under 380 nm excitation (Sun et al., 2016). The
absorption spectrum of oxOPD overlapped well with the emission spectrum of CDs,
which was a prerequisite for the IFE (Fig. S6A). After incubation of CDs with
oxOPD, the fluorescence intensity of CDs was efficiently quenched (Fig. S6B).
Meanwhile, no evident change in the fluorescence lifetime of the CDs was observed
with oxOPD, indicating that there was no energy and/or electron transfer between
CDs and oxOPD (Fig. S6C). In addition, no significant changes in the absorption
spectra of oxOPD were observed with the addition of CDs, which indicated that there
was a lack of complex formation and no covalent bonding between CDs and oxOPD
7

(Fig. S6D). Meanwhile, the zeta potentials of CDs and oxOPD were all positively
charged, and oxOPD could not be adsorbed on the surface of CDs via electrostatic
interactions (Fig. S6E). Considering the above two points, the distance between CDs
and oxOPD was hardly shorter than 10 nm (Liu et al., 2017). Therefore, the
fluorescence resonance energy transfer process could not occur in this system, and the
fluorescence quenching mechanism of CDs and oxOPD was caused by the IFE. The
2
+
final concentration of Cu was chosen to be 70 μM in the subsequent experiments
due to the sufficient enhancement (Fig. S7).
3
.3 Sensing of AChE and BChE in buffer solution
There were no significant changes in the fluorescence spectra of the system with
individual substrates (ATCh or BTCh) or enzymes (AChE or BChE) (Fig. S8A).
2
+
However, when ATCh was used together with CDs, OPD, Cu , AChE or BChE, the
fluorescence intensity at 460 nm was recovered, while the fluorescence intensity at
5
70 nm disappeared (Fig. S8B, curves c and d). Additionally, when using BTCh as the
substrate, only BChE could lead to the above fluorescence spectrum change (Fig.
S8B, curve f). These results demonstrated that ATCh could be hydrolyzed by AChE
and BChE, while BTCh was preferentially hydrolyzed by BChE. Moreover, Etho was
a selective BChE inhibitor and did not inhibit AChE activity (Worek et al., 1999).
Therefore, by employing ATCh as the substrate together with Etho, or BTCh as the
substrate, the changed fluorescence spectra could verify the individual activity of
AChE or BChE given the coexistence of AChE and BChE (Fig. S8B, curves g and h).
These results indicated that the probe can be applied to discriminatively determine
AChE and BChE in human whole blood.
Under optimal conditions (Fig. S9), the fluorescence intensity at 460 nm
gradually increased, while the fluorescence intensity at 570 nm gradually decreased
8

with increasing AChE or BChE concentrations (Fig. 3A and B). Moreover, with the
increasing AChE or BChE concentrations, the photographs exhibited not only a naked
eye color change from yellow to colorless under visible light, but also a fluorescence
color change from yellow to blue under UV light. The AChE or BChE activity could
-1
-1
be determined in the range 0.2 − 14.0 U L or 0.1 − 5.0 U L with a linear range from
-1
-1
0
.2 to 4.0 U L or 0.1 to 1.2 U L (Fig. 3C and D). The linear relationship equations
2
2
were y = 0.038 x + 0.115 (r = 0.990) for AChE and y = 0.159 x + 0.111 (r = 0.992)
-1
for BChE. The detection limits of AChE and BChE were estimated to be 0.1 U L and
-
1
0
.04 U L according to 3σ rule. These values were comparable to or lower than those
of existing probes, and a detailed comparison is summarized in Table S1. Although
Ellman’s method had the advantages of convenience and accessibility of high-
throughput analysis (Ellman et al., 1961), the developed method provided excellent
selectivity and sensitivity for AChE and BChE assay and avoided the false-positive
signal, background interference, and pretreatment of whole blood. These results
demonstrated that the CDs-based ratiometric fluorescence probe was capable of the
highly sensitive detection of AChE and BChE.
9

Fig. 3. Fluorescence spectra of CDs-based probe after incubation with different
concentrations of (A) AChE and (B) BChE. Insets: corresponding photographs under
visible (top) and UV light (bottom). The curve of F /F (ratiometric fluorescence
4
60 570
intensity) versus the concentration of (C) AChE and (D) BChE. Insets: the linear
relationship between F /F and ChEs concentration.
4
60 570
We further investigated possible interferents including alkaline phosphatase
ALP), human serum albumin (HSA), glucose oxidase (GOx), thrombin, trypsin and
(
glutathione (GSH) for the specificity assay. Except for GSH, these species showed no
apparent interference with the determination of ChEs (Fig. S10). However, the
interference of GSH could be effectively eliminated by using the thiol scavenger N-
ethylmaleimide. Although AChE and BChE can both catalyze the hydrolysis of
ATCh, by employing ATCh as the substrate together with Etho, or BTCh as the
substrate, the probe could verify the individual activtiy of either AChE or BChE, even
1
0

in the coexistence of AChE and BChE. These results indicated that the probe can be
applied for the selective determination of AChE and BChE activtiies.
3
.4 Inhibition evaluation of AChE and BChE
Tacrine and Etho are well-known inhibitors of AChE and BChE, respectively.
The ratiometric fluorescence intensity (F /F ) gradually decreased with increasing
4
60 570
concentrations of tacrine and Etho, demonstrating the inhibition of AChE and BChE
catalyzed hydrolysis reactions (Fig. S11). The corresponding IC50 values of tacrine
and Etho were estimated to be 29.8 nM and 132.6 nM, which are comparable to or
lower than those existing ChEs assays (Li et al., 2013; Worek et al., 1999). These
results demonstrated that the CDs-based probe holded great potential for screening
ChEs inhibitors.
3
.5 Determination of AChE and BChE in human whole blood
The whole blood samples were finally diluted 3000-fold and 10000-fold for
AChE and BChE determinations, respectively. For the AChE analysis, ATCh was used
as the substrate and Etho used as a selective BChE inhibitor. For the BChE analysis,
BTCh was used as the substrate. The mean concentrations of GSH and cysteine in the
3
000-fold or 10000-fold diluted biological sample were diluted to the nanomolar
level. The additions of certain amounts of GSH, cysteine, α-lactalbumin, and β-
lactoglobulin exhibited negligible interference. In addition, the fluorescence responses
of CDs-based probe showed no clear changes among the different diluted biological
samples. As a result, the biological samples with adequate dilutions could circumvent
the background interference from GSH, cysteine, α-lactalbumin, and β-lactoglobulin
and did not affect the determination of AChE and BChE activities (Fig. S12). The
CDs-based probe could directly measure individual AChE and BChE activities in
whole blood, while the Ellman method could not detect whole blood AChE (Table
1
1

S2). The activities of AChE and BChE in whole blood were in agreement with those
previously reported ranges (Qian et al., 2016; Chen et al., 2018).
4
. Conclusions
We developed a ratiometric fluorescence probe based on CDs for highly sensitive
detection of AChE and BChE activities. Through the inhibition of the oxidization of
OPD by AChE or BChE catalyzed hydrolysis of ATCh or BTCh, the change in
ratiometric fluorescence intensity served as the signal output for ChEs activity sensing
and inhibitors screening. This method provided excellent selectivity and sensitivity
-
1
-1
for AChE and BChE assays with low detection limit of 0.1 U L and 0.04 U L ,
respectively. In addition, the proposed approach avoided complex modifications of
probe and expensive instrumentation. Moreover, by taking advantage of specific
substrates and Etho, the probe was capable of simultaneously and discriminatively
monitoring AChE and BChE in human whole blood without any pretreatment.
Importantly, the proposed method could circumvent the false-positive signal and
background interference in whole blood samples with adequate dilutions, indicating
the potential for application in clinical diagnosis. However, the work suffered the
limitation of not monitoring ChEs activities in living cells.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
(
No. 21705080 and 61775099), Natural Science Foundation of Jiangsu Province
BK20171043 and BK20171487), and Startup Foundation for Introduced Talents of
(
Nanjing Medical University (2016RC04).
1
2

Credit author statement
Xiaoman Xu and Yao Cen designed the study and performed major part of this
research including experiment, analysis and interpretation of data. Guanhong Xu,
Fangdi Wei and Menglan Shi helped to search literatures and to draw figures. Yao
Cen and Qin Hu supervised the whole project and wrote the manuscript. The authors
agree to be accountable for all aspects of the work. The final version has been
approved by all authors.
Declaration of interest statement
none
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Highlights
•
•
•
A ratiometric fluorescence probe based on carbon dots for cholinesterases sensing
Sensitive and selective cholinesterases activities sensing and inhibitors screening
Simultaneous and discriminative detection of cholinesterases in human whole blood
1
5